JMJD3 (KDM6B) is a chromatin regulator with roles central to normal development as well as a wide range of human diseases including cancer and human neurological disorders. For instance, mutations in JMJD3 are autosomal recessive for familial intellectual disability, and de novo JMJD3 mutations are associated with autism spectrum disorder (ASD). An important next step to understanding genetic causes of complex diseases is to study disease-associated genes in mouse models. While it is known that JMJD3 is important to certain aspects of neural cell development, whether JMJD3 deficiency can actually cause cognitive dysfunction has not been known. Preliminary Studies indicate that JMJD3 is critical for the development of the mouse hippocampal dentate gyrus (DG). In the DG, granule neurons are generated throughout life from a population of neural stem cells (NSCs). Defective DG neurogenesis impairs many hippocampal-dependent behaviors and has been associated with cognitive deficits including that of intellectual disability and ASD. Without Jmjd3, NSCs failed to become established in the adult DG, and granule neuron production was severely decreased and abnormal. In these mice, hippocampal-dependent learning was defective. Heterozygous deletion of Jmjd3 also resulted in abnormal postnatal DG development, indicating that this process is sensitive to gene dosage. Aim 1 is to determine the role of Jmjd3 in DG neurogenesis. In vivo experiments will test the hypothesis that Jmjd3 regulates the postnatal expansion and establishment of the DG NSC population, and that even reduced Jmjd3 gene dosage causes cognitive dysfunction. Single cell RNA sequencing analysis will provide molecular insights into the observed phenotype and help guide mechanistic studies of Aim 2. Aim 2 is to determine the mechanisms by which JMJD3 regulates gene expression. JMJD3 has demethylase activity for histone 3 lysine 27 trimethylation (H3K27me3), which is a chromatin modification associated with transcriptional repression. To investigate demethylase-dependent and potential demethylase-independent activities of JMJD3, we have developed innovative CRISPR-based technologies to recruit JMJD3 proteins to the genome. By developing a novel, easy-to-use method for mapping lamina-associated domains (LADs) ? a repressive nuclear compartment ? we have also found that JMJD3 in NSCs is enriched at the genomic LAD ?borders,? which are genomic regions enriched for transcriptional regulatory elements. Thus, we propose investigating the role of JMJD3 in regulating this aspect of higher-order chromatin structure. The proposed neurodevelopmental and behavioral analyses combined with mechanistic studies is expected to provide a scientific framework in which to begin understanding how human JMJD3 mutations can cause disease. The studies of JMJD3 mechanism is also expected to be important to the broader field of chromatin-based epigenetics as well as nuclear compartment-associated genome organization ? an emerging area of research.